Biology Reference
In-Depth Information
reactive oxygen in the algal medium (
Fischer et al., 2012
). A dedicated anal-
ysis of TrHb proteins in
Chlamydomonas
is clearly needed, and it is fascinating
that at least some TrHb proteins appear to be regulated in response to envi-
ronmental conditions in and around the cell.
5. IN VITRO CHARACTERIZATION
5.1. Structural properties
5.1.1 Nostoc commune GlbN
Electronic absorbance spectra provide a low-resolution assessment of
haemoglobin properties. The position, intensity and width of bands due
to the haem group are influenced by the oxidation state of the iron and
its axial ligands. The strongest absorption on the low energy side of
300 nm, generally appearing in the 300- to 500-nm range, is called the Soret
band. Lower intensity bands occur in the visible region (in the 500- to
700-nm range). Weak lower energy features appearing in the
600- to 700-nm range correspond to charge transfer (CT) transitions
observed in high-spin species. In contrast, aromatic residues (tryptophan,
tyrosine and phenylalanine) absorb in the near-UV (275-290 nm) and their
contribution can be roughly estimated based on amino acid composition.
The ratio of the Soret intensity at its maximum (
A
Soret max
) to that of the
maximum aromatic intensity (
A
aro max
) is often used to evaluate the degree
of haem incorporation in globins. Thus, optical spectra are well suited for
rapid preparative and comparative purposes.
For reference, it is useful to review the characteristics of sperm whale
Mb. The physiological state contains ferrous iron. In the absence of exog-
enous ligand (i.e. O
2
or CO), the ferrous state is a five-coordinate (5c) para-
magnetic species (four unpaired electrons,
S
2) referred to as the deoxy
state. Deoxymyoglobin can bind dioxygen (yielding a diamagnetic complex,
S
¼
1/2). In the ferric
state, also referred to as 'met', myoglobin contains either a hydroxide ion
or a water molecule as the sixth ligand to the iron. The aquomet complex
is 'high-spin' (five unpaired electrons,
S
¼
0), carbon monoxide (
S
¼
0) and nitric oxide (
S
¼
5/2), whereas the hydroxymet
complex exists as an
S¼
1/2,
S¼
5/2 spin equilibrium. The ionization of
the water ligand occurs with a p
K
a
of
¼
9, but the value depends strongly
on the protein (
Antonini & Brunori, 1971
). Cyanide is a strong-field ligand
to ferric iron, which leads to a low-spin (
S
1/2) complex. Cyanomet glo-
bins are generally stable and regularly used as an isoelectronic version of the
carbonmonoxy complex. Liganded azide and fluoride yield a mixed spin and
¼
